The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells

  1. Limei Zou
  2. Clara Moch
  3. Marc Graille
  4. Clément Chapat

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Abstract

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.

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    SciScore for 10.1101/2022.01.25.477753: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: The absence of mycoplasma contamination in cells was routinely tested.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP)
    anti-eIF4E2/4EHP
    suggested: None
    anti-DDX6
    suggested: (Proteintech Cat# 14632-1-AP, RRID:AB_2091264)
    anti-CNOT9
    suggested: (Proteintech Cat# 22503-1-AP, RRID:AB_11232413)
    anti-Flag
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-GIGYF2
    suggested: (Proteintech Cat# 24790-1-AP, RRID:AB_2879727)
    Experimental Models: Cell Lines
    SentencesResources
    Flp-In T-REx 293 cells (Thermo Fisher Scientific) were grown in similar conditions supplemented with 100μg/ml zeocin and 15μg/ml blasticidin.
    Flp-In T-REx 293
    suggested: RRID:CVCL_U427)
    The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    CRISPR-Cas9-mediated genome editing of HEK293 cells was performed according to Ran et al. (53).
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Recombinant DNA
    SentencesResources
    The cell line inducibly expressing Flag-NSP2 was generated by co-transfecting pcDNA5-FRT-TO-FH-Nsp2 (Addgene plasmid 157683) and pOG44 (Thermo Fisher Scientific) with a 1:10 ratio.
    pcDNA5-FRT-TO-FH-Nsp2
    suggested: RRID:Addgene_157683)
    pOG44
    suggested: None
    Truncated versions of NSP2 were generated by replacing the sequence encoding full-length NSP2 by PCR-amplified fragments at the BamHI-XhoI sites of the pcDNA5-FRT-TO-FH vector.
    pcDNA5-FRT-TO-FH
    suggested: None
    The mCherry2-NSP2 expressing plasmids were created by cloning a PCR-amplified NSP2 fragment into the mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 vectors (Addgene plasmid 54517) using EcoRI and BamHI restriction enzymes.
    mCherry2-NSP2
    suggested: None
    mCherry2-C1
    suggested: RRID:Addgene_54563)
    mCherry2-N1
    suggested: RRID:Addgene_54517)
    To generate the pCI-λNV5-GIGYF2 vector, a fragment containing the GIGYF2 sequence was obtained by PCR from the pcDNA4/TO/GFP-GIGYF2 vector (Addgene plasmid 141189) (34) and inserted into the pCI-λNV5 at the XhoI-NotI sites.
    pCI-λNV5-GIGYF2
    suggested: None
    pcDNA4/TO/GFP-GIGYF2
    suggested: RRID:Addgene_141189)
    pCI-λNV5
    suggested: None
    A similar strategy was used to generate the V5-tagged fragments of GIGYF2 using primers to isolate the following domains encompassing residues: 1-267; 257-495; 485-752; 742-1,085; 1,075-1,320. pCI-eGFP-GIGYF2 was generated by inserting the eGFP sequence, PCR-amplified from pEGFP-C1 (Clontech), at the NheI-XhoI sites of the pCI-Neo vector and then by adding the fragment encoding GIGYF2 at the XhoI-NotI sites.
    pCI-eGFP-GIGYF2
    suggested: None
    pEGFP-C1
    suggested: None
    The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.
    pCI-Neo
    suggested: RRID:Addgene_16574)
    To generate the pCI-λNV5-GW182SD vector, a fragment encoding the residues 1382-1690 was cloned by PCR as a XhoI-NotI fragment from the pFRT/TO/FLAG/HA-DEST TNRC6C vector (Addgene plasmid 19885) (52) and inserted into the pCI-λNV5 at the XhoI-NotI sites.
    pCI-λNV5-GW182SD
    suggested: None
    pFRT/TO/FLAG/HA-DEST TNRC6C
    suggested: RRID:Addgene_19885)
    For recombinant GST-fused protein expression, full-length 4EHP and GIGYF2742-1,085 coding sequences contained in PCR-amplified BamHI-NotI and XhoI-NotI fragments, respectively, were cloned into a pGEX-6P-1 vector (Amersham) in frame with the GST coding sequence.
    pGEX-6P-1
    suggested: None
    The pET-28b vector (EMD Biosciences) was used to express NSP2 as a recombinant protein fused to an His6 tag at the N-terminus.
    pET-28b
    suggested: RRID:Addgene_47327)
    To generate KO HEK293 cells, we transfected 700,000 cells with the pSpCas9(BB)-2A-Puro plasmid.
    pSpCas9 ( BB)-2A-Puro
    suggested: None
    For the reporter containing the 3’UTR of Ifnb1, 20 ng of psiCHECK2-RLuc-Ifnb1 3’ UTR reporter (31), or empty psiCHECK2 (Promega), was added along with 100 ng of vectors encoding Flag-NSP2 or Flag (empty vector).
    psiCHECK2
    suggested: RRID:Addgene_40763)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.01.25.477753v1 on bioRxiv